Contraction of the heart occurs through regulated interactions of the myofilament proteins in response to increasing intracellular calcium concentrations. In heart failure both calcium dynamics and the response of the myofilaments to calcium are altered. These alterations impair systolic and diastolic function and are potentially reversible. This proposal seeks to define specific mechanisms for the effects of variants of the key regulatory protein troponin I which contribute to heart failure and hypertrophy. The hypothesis of this proposal is that post translational or genetic variants cardiac troponin I modify its function and play a central role in heart failure and the response to increased heart rate and afterload. The long-range goal of this work is to understand the underlying molecular mechanism by which these variants alter cardiac function in order to design strategies to prevent or treat cardiac dysfunction. The dissection of the molecular pathophysiology of troponin I variants will be approached in a series of highly collaborative integrative studies focused on modeling of myofilament disease-related post translational or genetic variants changes in vivo in murine models and in human cardiomyocytes. To address these goals the following aims are proposed: 1. To determine the degree and impact of altered site-specific phosphorylation of troponin I in human heart failure by quantitative phosphoproteomics in human cardiomyocytes and expression of recombinant phosphorylation site mutants of troponin I in human cardiomyocytes 2. To determine whether phosphorylation of PKA sites of troponin I or phospholamban are the dominant contributor to the in vivo frequency dependent acceleration of relaxation (FDAR) and the relaxation response to afterload by use of interbred mouse models and 3. To address the hypothesis that an exon 5 sequence variant of troponin I, which occurs with a 3 % frequency in African-Americans, influences the response to acute and chronic afterload through the use of an in vivo murine model. This work should provide insight into the in vivo effects of specific myofilament troponin I variants which contribute to pathophysiology of heart failure and hypertrophy. In the long-term this will assist in the development of novel therapies which address the correction of myofilament defects. PUBLIC HEALTH RELEVANCE: Heart failure is a significant medical problem in the United States. This proposal will study troponin I, a protein which regulates contraction of the heart. The specific goals are to study the effect of genetic and protein modifications of this protein on heart function. In the long term this should assist in developing better treatments for heart failure.